title: Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells.
abstract: OBJECTIVE
  To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells.
  
  
  METHODS
  Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes.
  
  
  RESULTS
  Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium.
  
  
  CONCLUSIONS
  These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.
authors.affiliation: College of Optometry, Western University of Health Sciences, Pomona, CA, USA.
authors.forename: Chunming
authors.initials: C
authors.lastname: Liu
authors.forename: Tshering
authors.initials: T
authors.lastname: Sherpa
authors.forename: Michael D
authors.initials: MD
authors.lastname: Varnum
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003117:Color Vision Defects / Q000235:genetics*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics*; D004110:Diltiazem / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D008024:Ligands; D009154:Mutation / Q000235:genetics*; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D020011:Protective Agents / Q000494:pharmacology; D014981:Xenopus
chemical_list: D017304:Annexin A5; C415904:CNGB3 protein, human; C474799:CPT-cGMP; D054815:Cyclic Nucleotide-Gated Cation Channels; D008024:Ligands; D020011:Protective Agents; D004110:Diltiazem; D006152:Cyclic GMP; D002118:Calcium